Design of artificial genes for use as controls in gene expression analysis systems

Abstract

Method of producing controls for use in gene expression analysis systems such as macroarrays, real-time PCR, northern blots, SAGE and microarrays. The controls are generated either from near-random sequence of DNA, or from inter- or intragenic regions of a genome. Ten specific control sequences are also disclosed. Also presented are methods of using these controls, including as negative controls, positive controls, and as calibrators of a gene expression analysis system.

1 - 25 . (canceled)  
     
     
         26 . A control for use in a gene expression analysis system comprising: 
 a known amount of at least one DNA target generated from at least one intergenic or intronic region of genomic DNA from a known sequence; or    a known amount of at least one spike mRNA generated from DNA generated from said at least one intergenic or intronic region of genomic DNA from a known sequence.    
     
     
         27 . The control of  claim 26 , wherein said at least one DNA target or spike mRNA do not hybridize at high stringency with any DNA or mRNA from a source other than said at least one intergenic or intronic region of genomic DNA.  
     
     
         28 . The control of  claim 26 , wherein said at least one DNA target or spike mRNA is formulated at selected concentrations and ratios.  
     
     
         29 . The control of  claim 26 , wherein said at least one DNA target is addressably disposed upon a substrate.  
     
     
         30 . The control of  claim 26 , wherein said at least one spike mRNA is detectably labeled.  
     
     
         31 . The control of  claim 26 , wherein said at least one DNA target or spike mRNA is between 500 and 2000 nucleotides in length.  
     
     
         32 . The control of  claim 26 , wherein said at least one DNA target or spike mRNA is about 1000 nucleotides in length.  
     
     
         33 . The control of  claim 26 , wherein 
 (a) said at least one DNA is selected from the group consisting of 
 (i) SEQ ID Nos: 1-10;  
 (ii) at least 17 contiguous nucleotides of SEQ ID Nos: 1-10; and  
 (iii) a complete complement of the sequence set forth in (i) and (ii); or  
   (b) said at least one mRNA is transcribed from the group consisting of 
 (i) SEQ ID Nos: 11-20;  
 (ii) at least 17 contiguous nucleotides of SEQ ID Nos: 11-20; and  
 (iii) a complete complement of the sequence set forth in (i).  
   
     
     
         34 . A control for use in a gene expression analysis system, comprising: 
 (a) a known amount of at least one DNA target, generated from a random mechanically synthesized sequence of DNA; and    (b) a known amount of at least one spike mRNA generated from said at least one DNA target.    
     
     
         35 . The control of  claim 34 , wherein said at least one DNA target or spike mRNA do not hybridize at high stringency with any DNA or mRNA from a source other than said sequence of DNA or mRNA.  
     
     
         36 . The control of  claim 34 , wherein said at least one DNA target or spike mRNA is formulated at selected concentrations and ratios.  
     
     
         37 . The control of  claim 34 , wherein said at least one DNA target is addressably disposed upon a substrate.  
     
     
         38 . The control of  claim 34 , wherein said at least one spike mRNA is detectably labeled.  
     
     
         39 . A method of using a control as a negative control in a gene expression analysis system comprising: 
 adding a known amount of said control DNA of  claim 26 , to a gene expression analysis system as a control sample;    subjecting the sample to hybridization conditions in the absence of complementary labeled mRNA;    examining the control sample for the absence or presence of signal.    
     
     
         40 . A method of using controls in a gene expression analysis system comprising: 
 adding a known amount of said control DNA of  claim 26 , to a gene expression analysis system as a control sample;    subjecting the sample to hybridization conditions in the presence of a known amount of labeled complementary mRNA of  claim 26;     measuring the signal values for the labeled mRNA and determining the expression level of the DNA based on the measured signal value.    
     
     
         41 . A method of using controls as calibrators in a gene expression analysis system comprising: 
 adding a known amount of a said control containing known amounts of several DNAs of  claim 26 , to a gene expression analysis system as control samples;    subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of corresponding complementary labeled mRNAs of  claim 26 , each mRNA being at a different concentration;    measuring the signal values for the labeled mRNAs and constructing a dose-response or calibration curve based on the relationship between signal value and concentration of each mRNA.    
     
     
         42 . A method of using controls as calibrators for gene expression ratios in a two-color gene expression analysis system comprising: 
 adding a known amount of at least one of said controls containing a known amount of DNA of  claim 26 , to a two-color gene expression analysis system as control samples;    subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of two differently labeled corresponding complementary labeled mRNAs of  claim 26 , for each DNA sample present;    measuring the ratio of the signal values for the two differently labeled mRNAs and comparing the signal ratio to the ratio of concentrations of the two or more differently labeled mRNAs.    
     
     
         43 . A process of producing at least one control of  claim 26 , which process comprising: 
 selecting at least one intergenic or intronic region of genomic DNA from a known sequence;    designing primer pairs for said at least one intergenic or intronic region;    amplifying said at least one intergenic or intronic region of genomic DNA to generate corresponding double stranded DNA;    cloning said double stranded control DNA using a vector to obtain additional double stranded DNA; and    formulating at least one control comprising said double stranded DNA.    
     
     
         44 . The process of  claim 43 , further comprising: 
 testing said at least one intergenic or intronic region to ensure lack of hybridization with mRNA from sources other than said at least one intergenic or intronic region of genomic DNA.    
     
     
         45 . The process of  claim 43 , further comprising: 
 generating mRNA complementary to said double stranded control DNA; and    formulating at least one control comprising said mRNA.    
     
     
         46 . The process of  claim 45 , further comprising: 
 purifying said DNA and mRNA;    determining the concentrations thereof; and    formulating at least one control comprising said DNA or said mRNA at selected concentrations and ratios.    
     
     
         47 . A method of using a control as a negative control in a gene expression analysis system comprising: 
 adding a known amount of said control containing a known amount of DNA of  claim 34 , to a gene expression analysis system as a control sample;    subjecting the sample to hybridization conditions in the absence of complementary labeled mRNA;    examining the control sample for the absence or presence of signal.    
     
     
         48 . A method of using controls in a gene expression analysis system comprising: 
 adding a known amount of a said control containing a known amount of DNA of  claim 34 , to a gene expression analysis system as a control sample;    subjecting the sample to hybridization conditions in the presence of a known amount of labeled complementary mRNA of  claim 34;     measuring the signal values for the labeled mRNA and determining the expression level of the DNA based on the measured signal value.    
     
     
         49 . A method of using controls as calibrators in a gene expression analysis system comprising: 
 adding a known amount of a said control containing known amounts of several DNAs of  claim 34 , to a gene expression analysis system as control samples;    subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of corresponding complementary labeled mRNAs of  claim 34 , each mRNA being at a different concentration;    measuring the signal values for said labeled control mRNAs and constructing a dose-response or calibration curve based on the relationship between signal value and concentration of each mRNA    
     
     
         50 . A method of using controls as calibrators for gene expression ratios in a two-color gene expression analysis system comprising: 
 adding a known amount of at least one of said controls containing a known amount of DNA of  claim 34 , to a two-color gene expression analysis system as control samples;    subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of two differently labeled corresponding complementary labeled control mRNAs of  claim 34 , for each DNA sample present;    measuring the ratio of the signal values for the two differently labeled mRNAs and comparing the signal ratio to the ratio of concentrations of the two or more differently labeled mRNAs.    
     
     
         51 . A process of producing at least one control of  claim 34 , which process comprising: 
 synthesizing a near-random sequence of DNA;    designing primer pairs for said sequence of DNA;    amplifying said DNA to generate corresponding double stranded DNA;    cloning said double stranded control DNA using a vector to obtain additional double stranded DNA; and    formulating at least one control comprising said double stranded DNA.    
     
     
         52 . The process of  claim 51 , further comprising: 
 testing said DNA to ensure lack of hybridization with mRNA from sources other than said DNA.    
     
     
         53 . The process of  claim 51 , further comprising: 
 generating mRNA complementary to said DNA; and    formulating at least one control comprising said mRNA.    
     
     
         54 . The process of  claim 53 , further comprising: 
 purifying said DNA and mRNA;    determining the concentrations thereof; and    formulating at least one control comprising said DNA or said mRNA at selected concentrations and ratios.